Kinetoplastid parasites such as Trypanosoma brucei and Leishmania cause devastating diseases that afflict millions of people. Summary. The whip-like flagellum that mediates motility in these single-cell parasites plays a critical role in the disease causing stages of both parasites, bloodstream form (BF) T. brucei and amastigotes of Leishmania. Our laboratory has discovered a novel cytoskeletal-associated protein, KHARON1, which is associated with the base of the flagellar axoneme and mediates targeting of integral membrane proteins to the flagellar membrane (FM) in both L. mexicana and T. brucei. Although likely to be important for all kinetoplastid parasites, this application will focus on the functionof KHARON1 in T. brucei due to the many technical advantages of this model parasite. Knockdown of TbKharon1 mRNA is lethal to BF trypanosomes, where it prevents cell division or cytokinesis, probably because it impairs flagellar targeting of various integral membrane proteins and thus disrupts the well-established role of the flagellum in initiating formation of th cleavage furrow that is required for cytokinesis. One major aim of this application will be to comprehensively identify the cohort of FM proteins that require TbKHARON1 to traffic to the flagellum using quantitative mass spectrometry. Defining the KHARON-dependent FM proteome will be critical for understanding the biological function of this novel flagellar trafficking machinery. However, recent studies with T. brucei have established that TbKHARON1 is also located in the mitotic spindle during parasite division and is associated with the subpellicular cytoskeleton beneath the plasma membrane. These distinct locations suggest that TbKHARON1 may play multiple functions in the biology of the parasite. Preliminary data also indicate that KHARON1 is likely only one component of one or several multiprotein KHARON Complexes and that a subset of the protein subunits in these complexes may be specific for each of the 3 known subcellular locations of KHARON1: the base of the flagellar axoneme, the mitotic spindle, and the subpellicular cytoskeleton. Hence, a second major aim will be to identify other protein subunits that associate with TbKHARON1 and in particular to search for subunits that may be specific for each of the 3 subcellular loci. These experiments will employ a recent methodology for identifying the components of multisubunit protein complexes, `biotin proximity labeling' or `BioID'. Once such additional subunits have been identified, their biological function will be interrogated using conditional RNA interference (RNAi). In particular, if site-specific KHARON Complexes and subunits exist, RNAi will be applied to such unique subunits to illuminate the potentially distinct roles of TbKHARON1 at the base of the flagellum, in the mitotic spindle, and in the subpellicular cytoskeleton. Overall, this project will define the biological an molecular functions in T. brucei of KHARON1, a protein that plays essential roles for viability of the disease-causing stage of African trypanosomes and other kinetoplastid parasites.